Kappacin, the nonglycosylated, phosphorylated forms of bovine caseinomacropeptide (CMP), has been shown to have antibacterial activity in vitro against both Gram-negative and Gram-positive oral bacteria (Malkoski et al., 2001). CMP is a 64 amino acid polypeptide released from bovine κ-casein by chymosin hydrolysis of the peptide bond between Phe105 and Met106. It comprises the 106-169 C-terminal fragment of κ-casein and contains all the post-translational modification sites found in κ-casein. CMP is both variably phosphorylated and glycosylated (Pisano et al., 1984; Saito and Itoh, 1992; Talbo et al., 2001). CMP is completely phosphorylated at Ser149 and partially phosphorylated (10%) at Ser127 as determined by MALDI-PSD mass spectrometry (Talbo et al., 2001). Additionally there are at least six genetic variants of κ-casein, with variants A and B being by far the most common (Creamer and Harris, 1997). Variants A and B differ at residues 136 and 148where the hydrophilic residues Thr136 and Asp148 of variant A are substituted by the hydrophobic residues Ile136 and Ala148 in variant B. The antibacterial active region of Kappacin was demonstrated to be residues 138-158 as determined using the synthetic peptide Ser(P)149κ-casein-A(138-158). Phosphorylation of Ser149 was shown to be essential for antibacterial activity using the synthetic peptide κ-casein-A(138-158) (Malkoski et al., 2001). The MIC of CMP variant A against Streptococcus mutans 0.68 mg/ml (100 μM) whilst variant B was less active with a MIC of 1.04 mg/ml (154 μM) (Malkoski et al., 2001).
The mechanism by which Kappacin inhibits bacterial growth is still unclear. Kappacin was found to be most effective against S. mutans at slightly acidic growth pH. The non-glycosylated, κ-casein-B(130-158) has been proposed to form an amphipathic α-helix, especially in the presence of trifluoroethanol (TFE; Plowman, 1997). This characteristic could help to explain its antibacterial activity if it works as a surface-active agent, creating pores in the cell membrane. This mode of action has been proposed for the majority of the cationic antimicrobial peptides isolated to date. However Kappacin is an anionic peptide that does not exhibit sequence similarity with the better known cationic an tibacterial peptides and apart from a possible propensity to form an amphipathic helical structure does not posses any of the other characteristics of these peptides. Kappacin does share some characteristics with the recently discovered anionic antibacterial peptides, especially enkelytin. This peptide, like Kappacin, contains a number of glutamyl residues and phosphorylation is essential for antibacterial activity (Goumon, 1996; Goumon, 1998; Strub, 1996). The structure of the phosphorylated form of enkelytin has not been determined, although phosphorylation has been proposed to change the conformation of the peptide through electrostatic repulsion or by divalent metal ion binding (Goumon, 1998; Kieffer, 1998). It remains unclear how the negatively charged antibacterial peptides, including Kappacin, interact with the bacterial cell surface.